Stroke is the third leading cause of death and the most common cause of neurological disability in the US. Although much work has focused on damage to neurons in the brain, essentially none of the therapies developed from this perspective has proven efficacious in clinical trials. Recent work has highlighted the important and multifaceted roles that glial cells play both in normal brain and following insults like stroke. The long term goal is to understand both the beneficial and deleterious effects of astrocytes and microglia in stroke to identify new targets to reduce brain injury. Heat shock proteins of the 70 kDa family have been shown to be protective in animal models of stroke, but to date only a single member of this family, the cytosolic Hsp72, has been extensively studied. Brain injury following stroke is known to involve oxidative stress and calcium overload, both of which are implicated in damage to both mitochondria and the endoplasmic reticulum. Both of these organelles play key roles in determining whether brain cells can survive ischemic injury, because they are intimately involved in dealing with the calcium overload and the free radicals that are hallmarks of ischemic injury. The first aim of this proposal will study the extent to which two Hsp70 family members localized to these organelles, Hsp75 to mitochondria and Hsp78 to endoplasmic reticulum, protect the brain from focal ischemia and modulate glial activation. This aim includes studies of modulation of inflammation by alteration of mitochondrial metabolic function and oxidative stress. The second aim targets the contribution of individual glial cell types to stroke. The cytosolic Hsp72, which is protective when expressed in all cell types, will be targeted selectively to either astrocytes or microglia to determine if overexpression restricted to these cells is sufficient to protect the brain and preserve neurons. Activation of microglia and astrocytes is important in the inflammatory response to stroke, and Hsp72 overexpression has been shown to modulate this response and reduce injury. The effect on inflammation of targeted expression will be determined. Because many aspects of cell function are altered simultaneously during ischemia, it has been difficult, to date, to identify the key pathways and mechanisms that determine outcome. In the third aim a dynamical computational model of inflammatory and apoptotic cell death signaling pathways, including modulation by Hsp70, will be developed. This will allow the use of computational tools to identify critical control strategies used in the molecular signaling interactions triggered by stroke. The model will be validated by biological experiments, and then used to identify the regulatory strategies evoked in response to inflammatory and ischemic stress. This dynamical computational analysis will identify the points at which intervention is most likely to strongly influence outcome. By approaching the problem from several complementary angles, new candidates for brain protection will be identified. PUBLIC HEALTH RELEVANCE Stroke is the third leading cause of death and the most common cause of neurological disability in the US. The goal of this research is to identify new ways to reduce the brain injury caused by stroke which when translated to clinical use could improve the lives of stroke victims by reducing the damage caused by stroke.